While it is known that opioids exert a profound influence on immunomodulating activity and have a cofactor role in the immunopathogenesis of HIV-1 disease, the mechanism(s) of their action remains to be determined. There is little information available about the association of opioid use with elevated plasma levels of lipopolysaccharide (LPS), a major component of gram-negative bacteria cell walls and a likely cause of systemic immune activation in HIV infection [1]. Particularly, it is unknown about the effect of opioid use in the presence of LPS on cellular microRNA and gene expression in HIV-1-targeted immune cells such as CD4+ T cells, monocytes, and microglia. Thus, to determine changes of miRNAs and genes related HIV infection of CD4+ T cells, monocytes, and microglia exposed to opioids is the area of interest, which may help to identify previously unidentified mechanisms involved in pathogenesis of HIV disease. The rationale of this proposal is based on the recent reports, showing that increased levels of plasma LPS in the blood is a consequence of translocation of bacterial products from a leaky gut, which is likely responsible for the systemic immune activation in chronic HIV infection [1] and that substance abuse is associated with high plasma LPS [2]. In addition, it has been recently documented that intracellular anti-HIV cellular factors in CD4+ T cells and monocytes/macrophages have a critical role in suppression of HIV infection/replication [3, 4]. Therefore, we hypothesize that drugs of use such as heroin is associated with elevated plasma levels of LPS and that opioids (heroin or morphine) suppress intracellular anti- HIV innate immunity in the immune cells and CNS cells, promoting HIV infection. We propose three specific aims to address previously unrecognized impact and mechanisms by which opioids and /or LPS compromise host cell innate immunity, increasing the susceptibility of immune and the CNS cells to HIV-1 infection and injury. In aim 1, we will examine in vivo impact of heroin use on the expression of markers of microtranslocation as well as on miRNA/gene profile of CD4+ T cells and monocytes with focus on the JAK/STAT pathways. In aim 2, we will examine in vitro impact of morphine and/or LPS on the expression of miRNA profiles with focus on newly identified anti-HIV miRNAs in CD4+ T cells, monocytes and macrophages. We will determine the molecular mechanisms responsible for morphine/LPS-mediated actions on intracellular innate immunity and HIV infection. In aim 3, we will determine the impact of opioids and/or LPS on the expression of anti-HIV miRNAs in astrocytes and microglia. We hypothesize that morphine and/or LPS, through the suppression of innate immunity in the CNS cells (primary astrocytes, microgila, and neurons), increase the susceptibility of microglia and astrocytes to HIV infection. The proposed studies are highly significant and innovative, as we will address the novel questions of whether opioid use is positively associated elevated plasma LPS in Chinese heroin dependent subjects, and whether opioids and/or LPS suppress intracellular anti-HIV innate immunity in the immune and CNS cells, promoting HIV infection.